Three-port chamber for processing particles

ABSTRACT

Embodiments relate to systems, chambers, and methods for processing particles by washing, concentrating, and/or treating the particles. Some embodiments provide for the processing of cells that may be in a liquid medium (e.g., a liquid in which the cells were grown) and are processed by being washed and concentrated for later use in research or other therapies.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional application of and claims priority to,U.S. patent application Ser. No. 14/827,130, entitled PROCESSINGPARTICLES, filed on Aug. 14, 2015, which claims priority to U.S.Provisional Patent Application No. 62/037,515, filed Aug. 14, 2014,entitled CONCENTRATION OF CELLS, which is hereby incorporated byreference in its entirety as if set forth herein in full.

BACKGROUND

Several processes require the concentration of particles. Particles maybe suspended or carried in a first volume of liquid and for additionalprocessing, it may be useful to concentrate the particles into a secondvolume that is less than the first volume. For example, within thebiological sciences there exists a need for concentrating cells. Oneexample where this is necessary is during an apheresis procedure wherecellular components of blood, e.g., leukocytes, erythrocytes,thrombocytes, are separated/concentrated from other liquid componentssuch as plasma. Another example is the post processing of cells that maybe grown in a liquid medium for therapeutic or research purposes. Thecells may be separated/concentrated with respect to the liquid medium inwhich they are grown. The separation or concentration of the cells mustoccur without having a significant effect on their viability for lateruse.

Embodiments of the present invention have been made in light of theseand other considerations. However, the relatively specific problemsdiscussed above do not limit the applicability of the embodiments of thepresent invention.

SUMMARY

The summary is provided to introduce aspects of some embodiments of thepresent invention in a simplified form, and is not intended to identifykey or essential elements of the claimed invention, nor is it intendedto limit the scope of the claims.

Embodiments relate to apparatuses, systems, and methods for processingparticles by washing, concentrating, and or treating the particles. Someembodiments provide for a chamber that includes an entry port forintroducing a first liquid and particles into a volume of the chamber.The chamber also includes a first exit port, located below the entryport, for removing the particles from the volume of the chamber afterthe particles have been processed. In some embodiments, a liquid may beintroduced into the chamber volume through the first exit port in orderto perform some processing steps, e.g., washing of the particles. Asecond exit port of the chamber is located above the first exit port andis utilized for removing liquid from the volume of the chamber. Thechamber also includes a sloped surface that directs at least a portionof the particles, introduced into the volume, toward the first exitport.

Other embodiments relate to method of processing particles. The methodsmay include use of a chamber and provide for subjecting the chamber to acentrifugal field by rotating the chamber. A first volume of particlesand liquid may be introduced into a chamber volume through a first port.A second liquid may be introduced into the chamber volume through asecond port. In embodiments, the second port may be positioned in ahigher force region of the centrifugal field than the first port. Liquidmay be removed through a third port, which may be positioned in a lowerforce region of the centrifugal field than the first port to concentratecells in the chamber volume. After concentration, a second volume ofparticles and liquid may be removed through the second port. The secondvolume may include a concentrated amount of particles compared to thefirst volume.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting and non-exhaustive embodiments are described with referenceto the following figures.

FIG. 1 illustrates an embodiment of a separation system, which can beused in, or with, embodiments.

FIG. 2 illustrates a tubing and bag set for use in, or with, embodimentsof the present invention.

FIG. 3 illustrates another example of a tubing and bag set for use in,or with, embodiments.

FIG. 4 illustrates a liquid processing vessel and an embodiment of achamber that may be used in combination in some embodiments.

FIGS. 5A-D illustrate a chamber consistent with a first embodiment.

FIGS. 6A-D illustrate a chamber consistent with a second embodiment.

FIGS. 7A-D illustrate a chamber consistent with a third embodiment.

FIGS. 8A-D illustrate a chamber consistent with a fourth embodiment.

FIGS. 9A-D illustrate a chamber consistent with a fifth embodiment.

FIGS. 10A-E illustrate views of a chamber consistent with a sixthembodiment.

FIGS. 11A-E illustrate views of two parts of chamber 1000 according toembodiments.

FIGS. 12A-C illustrate views of a chamber with respect to a spinningcentrifuge according to embodiments.

FIG. 13 illustrates a flow chart illustrating the steps of processingparticles consistent with embodiment(s).

FIG. 14 illustrates a centrifugal field and chamber ports, with respectto the centrifugal field, consistent with an embodiment.

DETAILED DESCRIPTION

The principles of the present invention may be further understood byreference to the following detailed description and the embodimentsdepicted in the accompanying drawings. It should be understood thatalthough specific features are shown and described below with respect todetailed embodiments, the present invention is not limited to theembodiments described below.

Embodiments below may be described with respect to processing cells suchas by separating cells from other cells or liquid components,concentrating cells, and/or washing cells. However, this is done simplyfor illustrative purposes. It is noted that the embodiments are notlimited to the description below. The embodiments are intended for usein products, processes, devices, and systems that process organic orinorganic particles, particulates, agglomerates. Accordingly,embodiments are not limited to separation, concentration, or washing ofcells, e.g., cellular components in whole blood, but may be used toseparate any particle from any liquid.

FIG. 1 illustrates one embodiment of a separation system 100, which canbe used in, or with, embodiments. In some embodiments, separation system100 provides for a continuous whole blood separation process. In otherembodiments, separation system 100 provides for concentrating andwashing cells. In one embodiment, whole blood is withdrawn from a donorand is substantially, continuously provided to a separation device 104where the blood is separated into various components and at least one ofthese components is collected from the device 104. One or more of theseparated components may be either collected for subsequent use orreturned to the donor. In embodiments, blood is withdrawn from the donorand directed through a bag and tubing set 108, which includes a tubingcircuit 112, and a liquid processing vessel 116, which together define aclosed, sterile and disposable system. The set 108 is adapted to bemounted in the separation device 104. The separation device 104 includesa pump/valve/sensor assembly 120, which interfaces with the tubingcircuit 112, and a centrifuge assembly 124, which interfaces with theliquid processing vessel 116.

In another embodiment, a volume of cells in liquid (e.g., suspended orcarried in liquid) is withdrawn from a storage container and issubstantially, continuously provided to separation device 104 where thecells are collected from the device 104 after concentrating and/orwashing. Additional liquid from processing the volume of cells in liquidmay be discarded. In embodiments, a bag with the volume of cells andliquid may be directed through tubing set 108, which includes a tubingcircuit 112, and a liquid processing vessel 116, which together define aclosed, sterile and disposable system. The set 108 is adapted to bemounted in the separation device 104, as noted above.

Examples of separation systems that may be the basis of systems usedwith embodiments of the present invention, e.g., separation system 100,include the SPECTRA OPTIA® apheresis system, COBE® spectra apheresissystem, and the TRIMA ACCEL® automated blood collection system, allmanufactured by Terumo BCT, Inc. of Lakewood, Colo.

The centrifuge assembly 124 may include a channel 128 in a rotatablerotor assembly 128 (e.g., centrifuge), where the channel 128 may be usedto hold a liquid processing vessel, e.g., vessel 116. The rotor assembly132 may rotate to create a centrifugal field. The rotor assembly 132 maybe configured to hold a chamber used to separate, concentrate, and/orwash cells. In one example, when whole blood is processed, cellularcomponents of blood may be separated from each other and from liquidcomponents of blood. In other examples, a volume of liquid containingcells may be processed with centrifuge assembly 124 to concentrate thevolume of cells.

The liquid processing vessel 116 may be fitted within the channel 128.In one example, blood can flow substantially continuously from a donor,through the tubing circuit 112, and into the rotating liquid processingvessel 116. Within the liquid processing vessel 116, blood may beseparated into various blood component types and at least one of theseblood component types (e.g., white blood cells, platelets, plasma, orred blood cells) may be removed from the liquid processing vessel 116and further processed. Blood components that are not being retained forcollection or for therapeutic treatment (e.g., platelets and/or plasma)may also be removed from the liquid processing vessel 116 and returnedto the donor via the tubing circuit 112.

In another example, a relatively large volume of liquid containingparticles (e.g., cells) may be preprocessed using the liquid processingvessel 116. The volume of liquid and cells may be initially flowed intothe liquid processing vessel 116 from tubing circuit 112. Within theliquid processing vessel 116, at least a portion of the liquid may beseparated from the particles and removed from the liquid processingvessel 116 through tubing circuit 112. A portion of the cells and liquidmay be retained for further processing.

Various alternative systems (not shown) may also be used withembodiments of the present invention, including batch processing systemsor smaller scale batch or continuous separation systems.

Operation of the separation device 104 may be controlled by one or moreprocessors included therein, and may comprise a plurality of embeddedcomputer processors that are part of a computer system. The computersystem may also include components that allow a user to interface withthe computer system, including for example, memory and storage devices(RAM, ROM (e.g., CD-ROM, DVD), magnetic drives, optical drives, flashmemory,); communication/networking devices (e.g., wired such asmodems/network cards, or wireless such as Wi-Fi); input devices suchkeyboard(s), touch screen(s), camera(s), and/or microphone(s); andoutput device(s) such as display(s), and audio system(s). In order tointerface with an operator of the system 100, embodiments of theseparation device 104 may include a graphical user interface 136 (shownin FIG. 1) with a display that includes an interactive touch screen.

An embodiment of a tubing circuit that may be used with embodiments isshown in FIG. 2, and as shown may include a cassette 200 and a number oftubing/storage assemblies 202, 204, 206, 208, and 210. In addition,tubing loops 220, 222, 224, 226, and 228 may engage with peristalticpumps on a separation device, e.g., device 104, to pump fluids throughthe tubing/storage assemblies. The tubing circuit also includes chamber218.

In embodiments, the tubing circuit shown in FIG. 2 may be used toseparate whole blood into components. In embodiments, some componentsseparated from whole blood may be returned to a donor, stored in one ormore storage containers, or further processed. For example, whole bloodmay be circulated through tubing of the tubing circuit and into theliquid processing vessel 216, which is mounted on a rotor assembly(e.g., assembly 128). Chamber 218 may also be mounted on the rotorassembly.

In the liquid processing vessel 216, the blood may separate intocomponents. Some components may be returned to a donor while others maybe further processed. For example, chamber 218 may be used to furtherprocess (concentrate or wash components of whole blood). In oneembodiment, red blood cells separated from whole blood may be introducedinto chamber 218 and concentrated before being stored in a container,e.g., a bag. In other embodiments, the red blood cells may be washed ortreated inside chamber 218, in addition to being concentrated, beforebeing stored in a container. As another example, platelets may bedirected to chamber 218 where they may be further processed(concentrated, washed, treated, etc.) before being stored in acontainer. Examples of chambers that may be used as chamber 218 in someembodiments are described in greater detail below, including descriptionof some chamber designs that have entry ports and exit ports in specificlocations (FIGS. 5A-11E).

Another embodiment of a tubing circuit that may be used with embodimentsis shown in FIG. 3. The tubing circuit includes a cassette 300 and anumber of tubing/storage assemblies 304, 308, 312, 316, 320, and 324. Inaddition, tubing loops 328, 332, 336, 340, 344, and 348 may engage withperistaltic pumps to pump fluids through cassette 300 and thetubing/storage assemblies. Assembly 312 provides a vent container, e.g.,bag that allows air displaced by liquid flowing in the tubing circuit tobe discharged into the vent container. The tubing circuit also includesa chamber 352. Examples of chambers that may be used as chamber 352 insome embodiments are described in greater detail below (FIGS. 5A-11E).

In embodiments, the tubing circuit shown in FIG. 3 may be used toconcentrate a volume of particles, e.g., cells, in liquid. Inembodiments, the tubing circuit may be mounted on a separation device sothat chamber 352 is mounted on a rotor assembly (e.g., assembly 128 witha centrifuge), which rotates the chamber 352.

Initially, a container storing the volume of particles with liquid maybe attached to tubing assembly 316. The particles in liquid may flowthrough tubing in the tubing circuit and into chamber 352. As describedin greater detail below, some embodiments provide for specific chamberdesigns that have entry ports and exit ports in specific locations. Asthe chamber 352 rotates, it may separate some liquid from the particlesand the removed liquid may flow through tubing into a container, e.g., awaste bag in assembly 320.

In some embodiments, the particles, in addition to being concentrated,may be washed or treated. In these embodiments, a wash or treatmentliquid storage container may be attached to tubing assembly 308. Thewash or treating liquid may flow through tubing into chamber 352 to washor treat the particles after, or during, concentration. The used wash ortreatment fluid may flow out of chamber 352 and into waste bag assembly320.

After the particles have been processed (concentrated, washed, and/ortreated), they may flow from chamber 352 through tubing in the tubingcircuit and through tubing assembly 324. In some embodiments, a storagecontainer may be attached to tubing assembly 324 to store the processedparticles. In other embodiments, tubing assembly 324 may be attached toan inlet of other tubing sets that are used to further process theparticles.

In some embodiments, the tubing circuit shown in FIG. 3 may include someadditional components. For example, shown in FIG. 4 is a chamber 400(which may be similar to chamber 352), connected to a liquid processingvessel 404 (which may be similar to vessel 216). In some embodiments,the liquid processing vessel 404 may be used for preprocessing of avolume of particles in liquid prior to processing in chamber 400.

As one example, if the volume of liquid with particles is relativelylarge, or it is desirable to process the volume at a high flow rate, itmay not be possible to process the volume directly by chamber 400. Inthese embodiments, vessel 404 may be used in a pre-processing step thatallows larger volumes (or flow rates) of liquid to be processed. Forexample, a centrifuge assembly (e.g., assembly 124) may include achannel in a rotatable rotor assembly (e.g., centrifuge), where thechannel may be used to hold the liquid processing vessel 404. The rotorassembly may rotate to create a centrifugal field. The rotor assemblymay also be configured to hold chamber 400.

Liquid (with the particles) may flow into vessel 404 through port 408.As the liquid flows around the vessel 404, which is rotating, liquidwith the particles may separate from other liquid that does not includethe particles, creating a concentrated volume of particles and liquid.The separated liquid (without particles) may be directed to a waste bag,with the a more concentrated stream of liquid and particles flowing intochamber 400, where the particles may be further concentrated, washed,and/or treated.

FIGS. 2-4 illustrate some embodiments of tubing circuits with chambersand vessels that are consistent with embodiments of the presentinvention. In embodiments, the tubing circuits of FIGS. 2-4 can bedisposable so that they are used one time to process a volume of liquid,e.g., blood, cells in growing medium, cells in storage medium etc., andthen discarded. In other embodiments, the tubing circuits can bereposable (reused) or include one or more component(s) that arereposable.

It is noted that the present invention is not limited to the specifictubing circuit configurations shown in FIGS. 2-4 and described above.Other tubing assembly arrangements, including additional components notshown in FIGS. 2-4 may be utilized in embodiments. For example, in someembodiments a tubing circuit may combine features of FIG. 3 and FIG. 4and include additional tubing that provides flow paths to the variouscomponents.

Below in FIGS. 5A-11E are various views of chamber designs that areconsistent with embodiments of the present invention. It is noted thatalthough specific examples are provided, the present invention is notnecessarily limited to the features of any one of the embodiments. Theembodiments shown in FIGS. 5A-11E are provided to illustrate somefeatures and illustrate examples of some embodiments. The presentinvention is therefore not limited to any specific embodiment.

As described in greater detail below, any of the chambers 500, 600, 700,800, 900, and 1000 shown in FIGS. 5A-11C may be used in embodiments toprocess particles. In some embodiments, the chambers may be used toconcentrate, wash, and treat particles, such as cells. In otherembodiments, the chambers may be used in an elutriation process, whereparticles of different sizes may be separated and collected separatelyfrom a mixture of liquid and multi-sized particles, e.g., cells.

FIGS. 5A-D illustrate four views of a chamber that may be used inembodiments to concentrate, wash, and/or treat particles in a liquid,such as cells in a liquid medium. FIG. 5A illustrates a top plan view ofchamber 500; FIG. 5B illustrates a side plan view of chamber 500; FIG.5C illustrates a cross-sectional view of chamber 500; and FIG. 5Dillustrates a perspective view of chamber 500.

Chamber 500 includes, inter alia, three ports. It is noted that by port,it is meant a perforation, e.g., a hole that allows liquid, particles,or other material to enter or exit a volume. Although reference numeralsin the figures may point to different features of the chambers in thefigures, it is noted that the ports are intended to refer to aperforation or hole in a wall or tube where liquid or particles enter orleave the volume of a chamber. The ports may be in fluid communicationwith pathways that direct liquid, particles, or other material through,or from, the port. In the figures, a reference numeral for a port maypoint to a pathway in fluid communication with the port, but the actualport is the perforation through which particles and/or liquid flowthrough as they enter or exit the chamber volume. The reference numeralmay point to the pathway in order to avoid confusion by crowding itemsin the figures or if the port is not visible in the figure (e.g. FIGS.12 and 14 below). However, where possible, the reference numerals pointto the actual port.

As shown in FIG. 5C, chamber 500 includes port 504, which is an entryport that allows liquid and particles to be introduced into a volume 508and may be located near a first cross section of volume 508 (e.g., crosssection of chamber 500 through line AA in FIG. 5B). Port 512 is an exitport through which particles and some liquid are removed from volume 508and may be located in a lower portion of volume 508. In addition, port516 is an exit port through which liquid separated from the particles(in order to concentrate the particles in a smaller volume of liquid,wash the particles with the liquid, and/or treat the particles with theliquid) is removed. Port 516 may be located in a top portion of volume508.

Chamber 500 also includes a sloped surface 520 that directs at least aportion of particles in volume 508 toward exit port 512. As illustratedin FIG. 5C, sloped surface 520 slopes toward exit port 512. Inoperation, chamber 500 may be subjected to a force, e.g., gravity orcentrifugal force that generally moves particles toward exit port 512.Sloped surface 520 aids in directing particles toward exit port 512.

In addition to the features described above, chamber 500 also includestop wall 524, sloped wall 528, and four side walls 532, 536, 540, and544. These walls define volume 508, which as shown is a trapezoidalvolume with a rectangular cross section when sectioned along lines AA orBB (FIG. 5B).

One feature of chamber 500 is that it includes at least twocross-sectional areas (e.g., one taken at line AA and the other taken atline BB), where the first cross-sectional area is larger than the secondcross-sectional area. As can be readily understood from FIGS. 5A and 5Bthe cross-sectional area taken along line AA would be rectangular and beof a larger area than a cross-sectional area taken along line BB.Without being bound by theory, it is believed that in some embodiments,this feature may provide a funneling effect toward exit port 512.

As shown in FIGS. 5A-D, exit port 516 is located in top wall 524.Although port 516 is shown in a particular location, in otherembodiments, exit port 516 may be located along any portion of top wall524. In other embodiments, exit port 516 may be in a side wall such asside walls 532, 536, 540, or 544. In some embodiments, the location ofexit port 516 is not limited to any specific location, except that it islocated above exit port 512.

Similarly, entry port 504 is in sidewall 532 and is shown in aparticular location. However, in other embodiments, entry port 504 maybe located in another location in side wall 532. In other embodiments,entry port 504 may be in a different side wall such as side walls 536,540, or 544. In some embodiments, the location of entry port 504 is notlimited to any specific location, except that it is located above exitport 512.

Sloped surface 520 is provided by sloped wall 528. Additionally, aportion of exit port 512 may be in sloped wall 528. However, in otherembodiments, exit port 512 may be located in another location such as inside walls 532, 536, 540, or 544. In some embodiments, the location ofexit port 512 is not limited to any specific location, except that it islocated below entry port 504.

In some embodiments, processing of particles may provide for introducingliquid or other material through port 512. In these embodiments, port512 may in addition to serving as an exit port for particles, may serveas an entry point for the liquid or other material.

FIGS. 6A-D illustrate four views of another chamber that may be used inembodiments to concentrate, wash, and/or treat particles in a liquid,such as cells in a liquid medium. FIG. 6A illustrates a top plan view ofchamber 600; FIG. 6B illustrates a side plan view of chamber 600; FIG.6C illustrates a cross-sectional view of chamber 600; and FIG. 6Dillustrates a perspective view of chamber 600.

Chamber 600 includes, inter alia, three ports. As shown in FIG. 6C,chamber 600 includes port 604, which is an entry port that allows liquidand particles to be introduced into a volume 608 and may be located neara first cross section of volume 608 (e.g., cross section of chamber 600through line CC in FIG. 6B). Port 612 is an exit port through whichparticles and some liquid are removed from volume 608 and may be locatedin a lower portion of volume 608. In addition, port 616 is an exit portthrough which liquid separated from the particles (in order toconcentrate the particles in a smaller volume of liquid, wash theparticles with the liquid, and/or treat the particles with the liquid)is removed. Port 616 may be located in a top portion of volume 608.

Chamber 600 also includes a sloped surface 620 that directs at least aportion of particles in volume 608 toward exit port 612. As illustratedin FIG. 6C, sloped surface 620 slopes toward exit port 612. Inoperation, chamber 600 may be subjected to a force, e.g., gravity orcentrifugal force that generally moves particles toward exit port 612.Sloped surface 620 aids in directing particles toward exit port 612.

In addition to the features described above, chamber 600 also includestop wall 624, sloped wall 628, and three side walls 632, 640, and 644.These walls define volume 608, which as shown is a triangular volume,with a rectangular cross section when sectioned along lines CC or DD(FIG. 6B).

One feature of chamber 600 is that it includes at least twocross-sectional areas (e.g., one taken at line CC and the other taken atline DD), where the first cross-sectional area is larger than the secondcross-sectional area. As can be readily understood from FIGS. 6A and 6Bthe cross-sectional area taken along line CC would be rectangular and beof a larger area than a cross-sectional area taken along line DD.Without being bound by theory, it is believed that in some embodiments,this feature may provide a funneling effect toward exit port 612.

As shown in FIGS. 6A-D, exit port 616 is located in top wall 624.Although port 616 is shown in a particular location, in otherembodiments, exit port 616 may be located along any portion of top wall624. In other embodiments, exit port 616 may be in a side wall such aswalls 632, 640, and 644; or sloped wall 628. In some embodiments, thelocation of exit port 616 is not limited to any specific location,except that it is located above exit port 612.

Entry port 604 is located in down tube 648 and is shown in a particularlocation. However, in other embodiments, entry port 604 may be locatedin another location. For example, down tube 648 may be shorter orlonger, which would change the location of entry port 604. In otherembodiments, down tube 648 may extend from a different wall (not topwall 624), such as wall 628, 632, 640, or 644. In some embodiments, thelocation of entry port 604 is not limited to any specific location,except that it is located above exit port 612.

Sloped surface 620 is provided by sloped wall 628 and slopes toward exitport 612. Additionally, a portion of exit port 612 is in sloped wall628. However, in other embodiments, exit port 612 may be located inanother location such as in walls 632, 640, or 644. In some embodiments,the location of exit port 612 is not limited to any specific location,except that it is located below entry port 604.

In some embodiments, processing of particles may provide for introducingliquid or other material through port 612. In these embodiments, port612 may in addition to serving as an exit port for particles, may serveas an entry point for the liquid or other material.

FIGS. 7A-D illustrate four views of another chamber that may be used inembodiments to concentrate, wash, and/or treat particles in a liquid,such as cells in a liquid medium. FIG. 7A illustrates a top plan view ofchamber 700; FIG. 7B illustrates a side plan view of chamber 700; FIG.7C illustrates a cross-sectional view of chamber 700; and FIG. 7Dillustrates a perspective view of chamber 700.

Chamber 700 includes, inter alia, three ports. As shown in FIG. 7C,chamber 700 includes port 704, which is an entry port that allows liquidand particles to be introduced into a volume 708, and may be locatednear a first cross section of volume 708 (e.g., cross section of chamber700 through line EE in FIG. 7B). Port 712 is an exit port through whichparticles and some liquid are removed from volume 708 and may be locatedin a lower portion of volume 708. In addition, port 716 is an exit portthrough which liquid separated from the particles (in order toconcentrate the particles in a smaller volume of liquid, wash theparticles with the liquid, and/or treat the particles with the liquid)is removed. Port 716 may be located in a top portion of volume 708.

Chamber 700 also includes a sloped surface 720 that directs at least aportion of particles in volume 708 toward exit port 712. As illustratedin FIG. 7C, sloped surface 720 slopes toward exit port 712. Inoperation, chamber 700 may be subjected to a force, e.g., gravity orcentrifugal force that generally moves particles toward exit port 712.Sloped surface 720 aids in directing particles toward exit port 712.

In addition to the features described above, chamber 700 also includestop wall 724 and side wall 728. These walls define volume 708, which asshown is an elliptical, sloping volume, with an elliptical cross sectionperpendicular when sectioned along lines EE or FF (FIG. 7B).

One feature of chamber 700 is that it includes at least twocross-sectional areas (e.g., one taken at line EE and the other taken atline FF), where the first cross-sectional area is larger than the secondcross-sectional area. As can be readily understood from FIGS. 7A and 7B,the cross-sectional area taken along line EE would be elliptical and beof a larger area than a cross-sectional area taken along line FF.Without being bound by theory, it is believed that in some embodiments,this feature may provide a funneling effect toward exit port 712.

As shown in FIGS. 7A-D, exit port 716 is located in top wall 724.Although port 716 is shown in a particular location, in otherembodiments, exit port 716 may be located along any portion of top wall724. In other embodiments, exit port 716 may be in side wall 728. Insome embodiments, the location of exit port 716 is not limited to anyspecific location, except that it is located above exit port 712.

Entry port 704 is in sidewall 728 and is shown in a particular location.However, in other embodiments, entry port 704 may be located in anotherlocation in side wall 728. In other embodiments, entry port 704 may bein a down tube that may extend from side 728 or from top wall 724. Insome embodiments, the location of entry port 704 is not limited to anyspecific location, except that it is located above exit port 712.

Sloped surface 720 is provided by side wall 728 and slopes toward exitport 712. Additionally, a portion of exit port 712 may be in side wall728. However, in other embodiments, exit port 712 may be located inanother location in side wall 728. In some embodiments, the location ofexit port 712 is not limited to any specific location, except that it islocated below entry port 704.

In some embodiments, processing of particles may provide for introducingliquid or other material through port 712. In these embodiments, port712 may in addition to serving as an exit port for particles, may serveas an entry point for the liquid or other material.

FIGS. 8A-D illustrate four views of another chamber that may be used inembodiments to concentrate, wash, and/or treat particles in a liquid,such as cells in a liquid medium. FIG. 8A illustrates a top plan view ofchamber 800; FIG. 8B illustrates a side plan view of chamber 800; FIG.8C illustrates a cross-sectional view of chamber 800; and FIG. 8Dillustrates a perspective view of chamber 800.

Chamber 800 includes, inter alia, three ports. As shown in FIG. 8C,chamber 800 includes port 804, which is an entry port that allows liquidand particles to be introduced into a volume 808 and may be located in acenter portion of volume 808. Port 812 is an exit port through whichparticles and some liquid are removed from volume 808 and may be locatedin a lower portion of volume 808. In addition, port 816 is an exit portthrough which liquid separated from the particles (in order toconcentrate the particles in a smaller volume of liquid, wash theparticles with the liquid, and/or treat the particles with the liquid)is removed. Port 816 may be located in a top portion of volume 808.

Chamber 800 also includes a sloped surface 820 that directs at least aportion of particles in volume 808 toward exit port 812. As illustratedin FIG. 8C, sloped surface 820 slopes toward exit port 812. Inoperation, chamber 800 may be subjected to a force, e.g., gravity orcentrifugal force that generally moves particles toward exit port 812.Sloped surface 820 aids in directing particles toward exit port 812.

In addition to the features described above, chamber 800 also includesbottom wall 824 and side wall 828. These walls define volume 808, whichas shown is a conical volume.

As shown in FIGS. 8A-D, exit port 816 is located at least partially inside wall 828. Although port 816 is shown in a particular location, inother embodiments, exit port 816 may be located along any portion ofside wall 828. In some embodiments, the location of exit port 816 is notlimited to any specific location, except that it is located above exitport 812.

Entry port 804 is in sidewall 828 and is shown in a particular location.However, in other embodiments, entry port 804 may be located in anotherlocation in side wall 828. In other embodiments, entry port 804 may bein a down tube that may extend from side wall 828 or from bottom wall824. In some embodiments, the location of entry port 804 is not limitedto any specific location, except that it is located above port 812.

Sloped surface 820 is provided by side wall 828 and slopes toward exitport 812. In embodiments, exit port 812 may be located in anotherlocation, such as in side wall 828. In some embodiments, the location ofexit port 812 is not limited to any specific location, except that it islocated below entry port 804.

In some embodiments, processing of particles may provide for introducingliquid or other material through port 812. In these embodiments, port812 may in addition to serving as an exit port for particles, may serveas an entry point for the liquid or other material.

FIGS. 9A-D illustrate four views of yet another chamber that may be usedin embodiments to concentrate, wash, and/or treat particles in a liquid,such as cells in a liquid medium. FIG. 9A illustrates a top plan view ofchamber 900; FIG. 9B illustrates a side plan view of chamber 900; FIG.9C illustrates a cross-sectional view of chamber 900, and FIG. 9Dillustrates a perspective view of chamber 900.

Chamber 900 includes, inter alia, three ports. As shown in FIG. 9C,chamber 900 includes port 904, which is an entry port that allows liquidand particles to be introduced into a volume 908 and may be located neara first cross section of volume 908 (e.g., cross section of chamber 900through line GG in FIG. 9B). Port 912 is an exit port through whichparticles and some liquid are removed from volume 908 and may be locatedin a lower portion of volume 908. Port 912 may in embodiments also beused as an entry port to introduce a liquid into the volume 908, such asto wash particles in volume 908. In addition, port 916 is an exit portthrough which liquid separated from the particles (in order toconcentrate the particles in a smaller volume of liquid, wash theparticles with the liquid, and/or treat the particles with the liquid)is removed. Port 916 may be located in a top portion of volume 908.

Chamber 900 also includes a sloped surface 920 that directs at least aportion of particles in volume 908 toward exit port 912. As illustratedin FIG. 9C, sloped surface 920 slopes toward exit port 912. Inoperation, chamber 900 may be subjected to a force, e.g., gravity orcentrifugal force that generally moves particles toward exit port 912.Sloped surface 920 aids in directing particles toward exit port 912.

In addition to the features described above, chamber 900 also includestop wall 924 and side wall 928. These walls define volume 908, which asshown is a frusto-conical volume. As is shown, side wall 928 includes afirst portion 928A and a second portion 928B.

One feature of chamber 900 is that it includes at least twocross-sectional areas (one taken at line GG and the other taken at lineHH), where the first cross-sectional area is larger than the secondcross-sectional area. As can be readily understood from FIGS. 9A and 9B,the cross-sectional area taken along line GG would be circular and be ofa larger area than a cross-sectional area taken along line HH. Withoutbeing bound by theory, it is believed that in some embodiments, thisfeature may provide a funneling effect toward exit port 912.

As shown in FIGS. 9A-D, exit port 916 is located in top wall 924.Although port 916 is shown in a particular location, in otherembodiments, exit port 916 may be located along any portion of top wall924. In other embodiments, exit port 916 may be in side wall 928. Insome embodiments, the location of exit port 916 is not limited to anyspecific location, except that it is located above exit port 912.

Entry port 904 is in sidewall 928 and is shown in a particular location.However, in other embodiments, entry port 904 may be located in anotherlocation in side wall 928. In other embodiments, entry port 904 may bein a down tube that may extend from top wall 924 or from side wall 928.In some embodiments, the location of entry port 904 is not limited toany specific location, except that it is located above exit port 912.Indeed, as illustrated in FIG. 9C, entry port 904 is located slightlyabove exit port 916.

Sloped surface 920 is provided by first portion of side wall 928A andslopes toward exit port 912. In other embodiments, exit port 912 may belocated in another location, such as in a portion of side wall 928. Insome embodiments, the location of exit port 912 is not limited to anyspecific location, except that it is located below entry port 904.

As noted above, processing of particles may provide for introducingliquid or other material through port 912. In these embodiments, port912 may in addition to serving as an exit port for particles, may serveas an entry point for the liquid or other material.

FIGS. 10A-11E illustrate a chamber 1000 that may be used in otherembodiments for processing particles, e.g., concentrate, wash, and/ortreat particles in a liquid, such as cells in a liquid medium. Asdescribed in greater detail below, chamber 1000 includes some featuresthat are similar to features of chambers 500, 600, 700, 800, and 900described above. Chamber 1000 also includes some additional featuresthat may be useful for processing some particles.

FIG. 10A illustrates a perspective view of chamber 1000; FIG. 10Billustrates a top plan view of chamber 1000; FIG. 10C illustrates a sideelevation view of chamber 1000; FIG. 10D illustrates a cross-sectionalview of chamber 1000; and FIG. 10E is a zoomed-in cross-sectional viewaround a port 1004. In some embodiments, chambers consistent with thepresent invention, e.g., chamber 1000, may be made from a single piece,e.g., a 3-D printed piece.

In other embodiments, chambers may be made from more than one piece FIG.11A illustrates an exploded view of chamber 1000 showing that thechamber may be made from two pieces 1100A and 1100B. FIGS. 11B and 11Cillustrate a top view and a bottom view of the first piece 1100A. FIGS.11D and 11E illustrate a top view and a bottom view of the second piece1100B. Reference numerals of the features described above with respectto FIGS. 10A-10E are used to refer to the same or similar features inFIGS. 11A-11E.

Referring to FIGS. 10A-E, chamber 1000 includes, inter alia, threeports. As shown in FIG. 10D, chamber 1000 includes port 1004, which isan entry port that allows liquid and particles to be introduced into avolume 1008 and may be located near a first cross section of volume 1008(e.g., cross section of chamber through line KK in FIG. 10C). Port 1012is an exit port through which particles and some liquid are removed fromvolume 1008 and may be located in a lower portion of volume 1008. Port1012 may in embodiments also be used as an entry port to introduce aliquid into the volume 1008, such as to wash particles in volume 1008.In addition, port 1016 is an exit port through which liquid separatedfrom the particles (in order to concentrate the particles in a smallervolume of liquid, wash the particles with the liquid, and/or treat theparticles with the liquid) is removed. Port 1016 may be located in a topportion of chamber 1000.

Chamber 1000 also includes a sloped surface 1020 that directs at least aportion of particles in volume 1008 toward exit port 1012. Slopedsurface 1020 slopes toward exit port 1012. In operation, chamber 1000may be subjected to a force, e.g., gravity or centrifugal force thatgenerally moves particles toward exit port 1012. Sloped surface 1020aids in directing particles toward exit port 1012.

One additional feature on chamber 1000 is alignment aid 1068 on sidewall 1028 (FIG. 10C). In embodiments, chamber 1000 may be positioned ona centrifuge and alignment aid 1068 may be used to ensure that whenmounted on the centrifuge, chamber 1000 is in a specific orientation.

Chamber 1000 also includes side wall 1028. Side wall 1028 defines volume1008. Side wall 1028 is angled with respect to central axis 1010defining volume 1008 as generally conical in shape, with a circularcross section such as cross sections at line KK or LL (FIG. 10C).

Chamber 1000 also includes a second volume 1032 which is located abovevolume 1008. As illustrated in FIG. 10D, second volume 1032 is definedby a side wall 1036 and a top wall 1040. As shown in FIG. 10D, exit port1016 is located in top wall 1040.

In some embodiments, the addition of a second volume may improve theprocessing of particles. For example, in one embodiment of processingcells, the additional volume provided by second volume 1032 allows alarger number (e.g., volume) of cells to be processed in chamber 1000.The additional volume may also be useful in embodiments where the cellsmay be washed, so that the wash liquid, which may be introduced throughport 1012 has room to flow through the cell bed in the lower portion ofvolume 1008 and any cells carried into volume 1032 have time to settleback into volume 1008 instead of immediately exiting through port 1016.

Top wall 1040, which defines the second volume 1032, may in someembodiments be substantially perpendicular to central axis 1010, e.g.,horizontal. Although as illustrated in FIGS. 10A-E, top wall 1040 isangled toward exit port 1016. Without being bound by theory, it isbelieved that in some embodiments, angling top wall 1040 toward exitport 1016 may provide a funneling effect toward exit port 1016.

In some embodiments, the angle between top wall 1040 and central axis1010 (see angle 1088 in FIG. 10C) may be between about 90 degrees (e.g.,top wall is horizontal) to about 5 degrees (e.g., top wall is steeplyangled toward port 1016). In other embodiments, the angle between topwall 1040 and central axis 1010 may be between about 90 degrees andabout 10 degrees, such as between about 80 degrees and about 15 degrees,or even between about 75 degrees and about 20 degrees.

Although port 1016 is shown in a particular location, e.g., collinearwith the central axis 1010, in other embodiments, exit port 1016 may belocated along any portion of top wall 1040. In other embodiments, exitport 1016 may be in side wall 1036. In some embodiments, the location ofexit port 1016 is not limited to any specific location, except that itis located above exit port 1012.

Entry port 1004 is in sidewall 1028 and is shown in a particularlocation. However, in other embodiments, entry port 1004 may be locatedin another location in side walls 1028 or 1036. In other embodiments,entry port 1004 may be in a down tube that may extend from side wall1028, side wall 1036, or top wall 1040. In some embodiments, thelocation of entry port 1004 is not limited to any specific location,except that it is located above exit port 1012.

Sloped surface 1020 is provided by side wall 1028 and slopes toward exitport 1012. Additionally, a portion of exit port 1012 is inside wall1028. However, in other embodiments, exit port 1012 may be located inanother location in side wall 1028. In some embodiments, the location ofexit port 1012 is not limited to any specific location, except that itis located below entry port 1004.

FIG. 10E illustrates a zoomed-in cross-sectional view of the area nearport 1004. This area includes a number of features that may be used insome embodiments. For example, a downward projecting baffle, namely wall1056, is positioned adjacent port 1004. In embodiments, when particlesin liquid are introduced through port 1004, wall 1056 directs flow ofthe particles in liquid toward an inside surface (e.g., surface 1020) ofside wall 1028. In some embodiments, wall 1056 may be curved or angledto further direct flow toward the inside surface of side wall 1028.

In addition, chamber 1000 may also include a second downward projectingbaffle, namely wall 1060 (FIG. 10D). As shown in FIG. 10D at least aportion of wall 1060 is located diametrically opposite wall 1056. Inembodiments, wall 1060 may prevent flow of particles in liquid intovolume 1032. For example, particles and liquid introduced through port1004 may create a flow (e.g., a jet) that causes particles already involume 1008 to flow into volume 1032. Wall 1060 at least partiallyprevents this from occurring. In some embodiments, wall 1056 and 1060may be part of a continuous circular skirt 1076 (see FIG. 11C).

Referring again to FIG. 10E, a channel 1044 is provided that is in fluidcommunication with the first port 1004. The channel 1044 may be at leastpartially defined by wall 1056. In embodiments, the channel 1044 directsflow of particles and liquid away from first port 1004.

In embodiments, the channel 1044 may direct flow of particles away fromfirst port 1004 in more than one direction. For example, the channel1044 may direct flow around wall 1056 in two directions. In embodiments,a partial wall 1048 may be positioned to only allow flow of particlesand liquid in one direction, e.g., in FIG. 10E out from the drawingsheet. FIG. 11C provides a different view of these features, namelychannel 1044, partial wall 1048, and wall 1056 and additionaldescription is provided below.

In some embodiments, first port 1004 may be in fluid communication withan entry pathway 1064 (FIGS. 10D and 10E) that may direct flow ofparticles and liquid to first port 1004. As may be appreciated, theentry pathway may direct flow from any direction to first port 1004. Inone embodiment, the entry pathway may direct flow from a directiongenerally parallel to central axis 1010 to port 1004 (see, e.g., FIGS.10D and 10E). In embodiments, port 1004 may include a sloped surface1052 to direct flow from the entry pathway toward wall 1056 and/orchannel 1044.

As noted above, some embodiments of chambers may be made from more thanone piece, and may be attached together to form a chamber. FIG. 11Aillustrates an exploded view of chamber 1000 showing that the chambermay be made from two pieces 1100A and 1100B. In embodiments, each ofpieces 1100A and 1100B may be manufactured separately, e.g., molded,milled, printed, etc. and attached together using any suitable process,e.g., adhered, welded, connected by fasteners, etc. Reference numeralsof the features described above with respect to FIGS. 10A-10E are usedto refer to the same or similar features in FIGS. 11A-11E.

FIGS. 11B and 11C illustrate a top view and a bottom view, respectively,of the first piece 1100A. FIG. 11B provides a similar view as the planview illustrated in FIG. 10B, showing top wall 1040, side wall 1036, andentry pathway 1064. FIG. 11C (bottom view of piece 1100A) illustrates adifferent view of channel 1044, partial wall 1048, wall 1056, and wall1066 than shown in FIG. 10E.

As illustrated in FIG. 11C, wall 1056 acts as a downward projectingbaffle and is adjacent to port 1004. In the embodiment illustrated inFIG. 11C, the wall 1056 is part of a skirt 1076 which extends in acircular shape and also provides wall 1060, a second downwardlyprojecting baffle, which is located diametrically opposite wall 1056. Inother embodiments, walls 1056 and 1060 may be separate features and notbe part of the continuous skirt 1076.

As shown in FIG. 11C, partial wall 1048 directs flow of particles andliquid that flow through port 1004 into channel 1044. Wall 1056, andskirt 1076, at least partially define channel 1044, which directs flowof liquid in the direction indicated by arrow 1072. In embodiments,channel 1044 directs flow of particles and liquid in a directiontangential to a sloped surface of a side wall (e.g., sloped surface 1020provided by side wall 1028). It is noted that although channel 1044 isillustrated as directing flow in one direction, in other embodiments,partial wall 1048 may be eliminated and flow of particles and liquid maybe allowed to proceed in more than one direction, e.g., at least in thedirection illustrated by arrow 1072 and in the direction illustrated byarrow 1080. FIG. 11C also illustrates exit port 1016.

FIGS. 11D and 11E illustrate a top view and a bottom view, respectively,of the second piece 1100B. FIG. 11D illustrates the sloped surface 1020provided by wall 1028, which slopes toward exit port 1012. Additionally,piece 1100B includes sloped surface 1052, which may be part of port1004. As discussed above with respect to FIG. 10E, some embodimentsprovide for an entry pathway (e.g., 1064) to direct fluid to port 1004.When fluid is being directed by an entry pathway from a directionsubstantially parallel to central axis 1010 (FIGS. 10C and 10D) a slopedsurface such as sloped surface 1052 may be used to redirect the flowtoward wall 1056 and into channel 1044.

As previously described, the embodiments illustrated in FIGS. 5A-11E areprovided merely for illustrative purposes. Embodiments are notnecessarily limited to the structural features shown in FIGS. 5A-11E anddescribed above. Other embodiments may include some features of theillustrated chambers and not others.

Some embodiments may include features consistent with features of somechambers shown in FIGS. 5A-11E but may not necessarily be describedabove. For example, some embodiments may provide chambers that include avolume with at least two cross-sectional areas; a second cross-sectionalarea being smaller than a first cross-sectional area. This embodimentmay include some of the chambers illustrated in FIGS. 5A-11E. In otherembodiments, the chamber may further provide for having an entry portnext to the first cross-sectional area and an exit port (e.g., forparticles) next to the second cross-sectional area, which are featuresof chambers 500, 600, 700, 900 and 1000. This is merely one example, anddifferent embodiments may include other features that may not benecessarily described above but still be within the scope of the presentinvention.

In addition to systems, tubing circuits, and chambers, some embodimentsrelate to methods of processing particles, such as cells, toconcentrate, wash, and/or treat particles. FIGS. 12-14 describe featuresof some methods consistent with embodiments. It is noted that althoughsome features of systems, tubing circuits, and chambers, may bementioned in the description below, they are provided merely forillustrative purposes and the methods are not necessarily limited tobeing performed by particular chambers, systems, or tubing circuits orother structural features, but rather may be performed by the structuresdescribed above or in other embodiments by other structures.

FIG. 12 illustrates a system 1200 that includes a centrifuge 1204. Thecentrifuge 1204 is configured to hold chamber 1208, which as describedin detail below may be used in processing particles in a liquid.Centrifuge 1204 rotates about an axis of rotation 1224. When centrifuge1204 holds chamber 1208, the chamber is rotated. As illustrated byarrows 1240 and 1244, embodiments provide for centrifuge 1204 to rotateclockwise (arrow 1240) or counterclockwise (arrow 1244).

Chamber 1208 may have any suitable design, some of which are describedabove in FIGS. 5A-11E. In the embodiment shown in FIG. 12, chamber 1208includes three ports, port 1212, port 1216, and port 1220. Port 1212 mayin embodiments be an inlet for introducing particles and liquid into avolume of chamber 1208, with ports 1216 and 1220 being outlets. Althoughreference numerals 1212, 1216, and 1220 point to walls of chamber 1208,it is noted that the ports are perforations or holes in the wall whereliquid or particles enter or leave the volume of chamber 1208. In theembodiments shown in FIG. 12, ports 1216 and 1220 are aligned withcentral axis 1222.

In some embodiments, methods provide for the position of one or moreports 1212, 1216, and/or 1220 during operation to be determined based onthe rotation of chamber 1208 when mounted and rotating on centrifuge1204, as is described in greater detail below.

FIG. 12B illustrates a view of chamber 1208, generally from thedirection illustrated by arrow 1236 (FIG. 12A). As shown in FIG. 12B,chamber 1208 is rotating clockwise as illustrated by arrow 1240. Alsoshown in FIG. 12B is plane 1252, which bisects chamber 1208 into a firstvolume 1256A and a second volume 1256B.

Similarly, FIG. 12C also illustrates a view of chamber 1208, generallyfrom the direction illustrated by arrow 1236 (FIG. 12A). FIG. 12C showschamber 1208 rotating counterclockwise as illustrated by arrow 1244.Also shown in FIG. 12C is plane 1252, which bisects chamber 1208 into afirst volume 1256A and a second volume 1256B.

Some embodiments provide for entry port 1212 to be in a particularlocation depending on the rotation of chamber 1208. In some embodiments,entry port 1212 may be located in a portion of a wall of chamber 1208that defines a trailing or leading volume, which is explained below.

In FIG. 12B, chamber 1208 is rotating clockwise, which makes thetrailing volume of chamber 1208 the second portion 1256B, and the firstportion 1256A is the leading volume. In embodiments where port 1212 maybe located on a portion of the wall defining a trailing volume, port1212 may be in a portion of the wall that defines volume 1256B, as shownin FIG. 12B.

In contrast, when chamber 1208 is rotating counterclockwise, such as inFIG. 12C, first volume 1256A is the trailing volume and second volume1256B is the leading volume. In embodiments where port 1212 may belocated on a portion of the wall defining a trailing volume, port 1212may be in a portion of the wall that defines volume 1256A, as shown inFIG. 12C.

It is noted that positioning port 1212 on a portion of the wall thatdefines the trailing or leading volume may be done using a variety ofmethods. In one embodiment, when chamber 1208 is being mounted, it maybe mounted in a position to ensure that entry port 1212 is in a portionof the wall defining the trailing or leading volume, whichever may bedesired. The position of port 1212 may be changed merely by rotatingchamber 1208 when mounting chamber 1208 on centrifuge 1204.

In other embodiments, chamber 1208 may be manufactured specifically forbeing rotated in a predetermined direction, e.g., clockwise orcounterclockwise. In these embodiments, chamber 1208 may be manufacturedwith the port 1212 positioned in the appropriate portion of the wall ofchamber 1208, that defines a trailing or leading volume, when chamber1208 is mounted on centrifuge 1204 and rotated.

It should be noted that although port 1212 is illustrated in aparticular location along the wall defining the trailing volume, it isnot limited to the locations shown in FIGS. 12B and 12C. That is, port1212 may be along any portion of the wall defining the trailing volume.For example, in FIG. 12B, port 1212 may be in any portion of the walldefining the trailing volume from a 6 o'clock position to a 12 o'clockposition, e.g., 7 o'clock, 8 o'clock, 9 o'clock, 10 o'clock etc.Similarly, in FIG. 12C, port 1212 may be in any portion of the walldefining the trailing volume from a 12 o'clock position to a 6 o'clockposition, e.g., 1 o'clock, 2 o'clock, 3 o'clock, 4 o'clock etc.

FIG. 13 illustrates a flow chart 1300 which may be performed inembodiments of the present invention. Although specific components maybe described below for performing steps in flow chart 1300, the presentinvention is not limited thereto. For example, some steps may bedescribed as performed by portions of system 1200, which as noted abovemay be implemented using system, chambers, tubing circuits, shown inFIGS. 1-11E. This is done merely for illustrative purposes, because flowchart 1300 is not limited to being performed by any specific components,structures, systems, apparatuses, chambers, or combinations thereof.

Flow 1300 begins at step 1304. Flow 1300 then passes to optional step1308, where a chamber may be oriented. As described above, embodimentsprovide for chambers (that may include three ports) for processing astream of particles and liquid, e.g., cells in a liquid medium. Inembodiments, the chambers may be mounted on different separation systemsthat may include a centrifuge. In embodiments, optional step 1308 may beperformed as part of mounting of a chamber on a separation system (e.g.,100), for example a centrifuge of a separation system. In otherembodiments, the chamber may be part of a disposable tubing circuit.Optional step 1308 may be performed as part of mounting the disposabletubing circuit onto a separation system, e.g., a centrifuge of aseparation system.

At step 1312, the chamber may be subjected to a centrifugal field. Insome embodiments, step 1312 may include one or more optional sub-stepsthat are performed as part of step 1312. For example, in someembodiments, a centrifugal field may be created by rotating the chamberat sub-step 1316. In embodiments, the rotation may be performed by partsof a separation or collection system, e.g., a centrifuge.

Referring to FIG. 14, an environment 1400 is illustrated that includes achamber 1404 being subjected to a centrifugal field 1408. Inembodiments, the centrifugal field 1408 may be generated by rotatingchamber 1404 around axis of rotation 1424. As illustrated by the size ofcircles 1412, 1416, and 1420, higher gravitational forces act on anobject (e.g., chamber 1404 or particles in chamber 1404) as they getfurther away from the axis of rotation 1424. As illustrated in FIG. 14,chamber 1404 also includes three ports 1432, 1436, and 1440. Inembodiments, chamber 1404 may have any suitable design, some of whichare described above in FIGS. 5A-11E.

After step 1312, flow 1300 may pass to optional step 1320 whereparticles in liquid may be pre-processed. The pre-processing step may beused to initially prepare a stream or volume of particles in liquid tobe processed by a chamber, such as chamber 1404.

In one embodiment, a first volume of particles in liquid may bepre-processed to reduce a volume in which the particles are carriedbefore the volume is processed in a chamber. For example, in someembodiments, a chamber may be connected to a liquid processing vessel(see liquid processing vessel 404 in FIG. 4). In embodiments, thepre-processing may involve introducing a first volume of particles andliquid into the liquid processing vessel and removing some liquid toreduce the volume.

In other embodiments, the pre-processing may involve introducing othermaterial into a volume of particles and liquid. The other materials maybe, for example, modifiers for the particles, liquid, or other substancein the chamber volume. For example, embodiments may involvepre-processing a volume of particles and liquid by adding substancesthat change a viscosity, density, and/or temperature of the volume. Inother embodiments, the other material may be added to modify theparticles, e.g., protect or change the particles before they undergoadditional processing. The foregoing are merely some examples ofpre-processing steps that may be performed in some embodiments.

Flow 1300 passes to step 1324 where a first volume of particles andliquid are introduced into a volume of a chamber through a first port.It is noted that in embodiments, step 1324 provides for introducingparticles and liquid through a first port, which has a specificlocation. For example, in one embodiment, step 1324 may involveintroducing the particles and liquid through a first port in a side wallthat defines a trailing volume of the chamber volume. Referring back toFIGS. 12A-12C, in these embodiments, as part of step 1324, a first port,e.g., entry port 1212 may be located in walls that define volumes 1256Aor 1256B, depending on the direction of rotation.

In another embodiment, the location of the first port may be in relationto the other ports and the centrifugal field to which the chamber issubjected. For example, step 1324 may involve introducing the particlesand liquid through a first port located in a higher force region than asecond port, but the first port may be in a lower force region than athird port, e.g., port 1436 (FIG. 14).

At step 1328, a second liquid may be introduced into a chamber volumethrough a second port, which in embodiments is located in a part of thechamber being subjected to higher forces from the centrifugal field.Referring to FIG. 14, step 1324 may involve adding the volume ofparticles in liquid through port 1432. Step 1328 may involve addingliquid through port 1440, which as shown in FIG. 14, is located in ahigher force region than port 1432.

In embodiments, the flow of the second liquid into the chamber mayprovide a specific function. For example, in embodiments, as the volumeof particles and liquid is introduced into the chamber volume, theparticles may begin to settle toward a higher force region see circle1420 (FIG. 14). In embodiments, step 1328 may be performed to preventthe particles from packing and or agglomerating to a point that theycannot be separated or are permanently damaged. The introduction of thesecond liquid may also maintain the particles at least partially in asuspension.

In other embodiments, the second liquid may be added to treat theparticles. For example, the second liquid may include some functionalgroup that may change the surface chemistry of the particles. In anotherembodiment, the second liquid may be designed to remove functionalgroups to change the surface chemistry. In yet other embodiments, thesecond liquid may provide some predetermined environment for theparticles.

It is noted that depending on the particles, the centrifugal field, thevolume of the chamber, and other factors, the flow rate of the secondliquid through the second port may vary. For example, in embodimentswhere the particles may be cells, the flow rate may be between about 1ml/min to about 50 ml/min, such as about 2 ml/min to about 45 ml/min,about 3 ml/min to about 40 ml/min, about 4 ml/min to about 35 ml/min, oreven about 5 ml/min to about 30 ml/min. In other embodiments, the flowrate of second liquid through second port may be less than about 35ml/min, less than about 30 ml/min, less than about 25 ml/min, less thanabout 20 ml/min, less than about 15 ml/min, less than about 10 ml/min oreven less than about 5 ml/min. In other embodiments, the flow rate ofsecond liquid through second port may be greater than about 2 ml/min,greater than about 4 ml/min, greater than about 6 ml/min, greater thanabout 8 ml/min, greater than about 10 ml/min, greater than about 12ml/min or even greater than about 14 ml/min.

After step 1328 flow 1300 passes to optional step 1332 where theviscosity of the liquid and/or particles in the chamber may be modified.Optional step 1332 may be performed in some embodiments as part ofprocessing the particles after they have been introduced into thechamber. For example, in some embodiments, the particles may be washedor treated in the chamber. Optional step 1332 may be performed as partof the process of washing and/or treating the particles after the volumeof particles to be processed have been introduced into the chamber.

In some embodiments, optional step 1332 may involve one or moresub-steps. For example, sub-step 1336 may be performed to add secondliquid through the first port. Referring to FIG. 14, sub-step 1336 mayinvolve adding the second liquid through port 1432. In some embodiments,after the viscosity has been changed at step 1332, the flow of liquidthrough the first port may be reduced to a rate lower than the rateinitially used to change the viscosity.

In other embodiments, the viscosity change performed by step 1332 may beeffected by a temperature change. The temperature change may occur incombination with addition of a liquid, e.g., sub-step 1336. For example,the second liquid introduced through the first port at sub-step 1336 maybe at a predetermined temperature that allows the temperature of theparticles and liquid in the chamber to be controlled. In otherembodiments, step 1332 may simply involve the control of the temperatureof the liquid and particles in the chamber, such as through conduction,convection, or radiation. In yet other embodiments, the second liquidmay simply displace liquid already in the volume and thus change theviscosity.

Flow 1300 passes to optional step 1340 where particles in the chambermay be washed. Optional step 1340 may be performed to replace the liquidthat entered the chamber with the particles, with a different liquid.Referring to FIG. 14, step 1340 may involve increasing the flow ofsecond liquid through port 1440.

In other embodiments, optional step 1340 may be performed to, inaddition to replacing liquid, also remove a material from the particlesor the liquid. For example, in embodiments where the particles may becells, the cells may initially be in a liquid medium that includes avariety of proteins, nutrients, and waste materials. Optional step 1340may be used to remove these materials from the cells before the cellsmay be used for research or therapeutic purposes.

Optional step 1340 may in embodiments involve one or more sub-steps. Forexample, in embodiments, sub-step 1344 may be performed to increase theflow rate of the second liquid through the second port. In embodiments,the additional flow rate of liquid will flow through the particles thathave previously been maintained in suspension by the flow rate of liquidthrough the second port.

Depending on the particles, the centrifugal field, the volume of thechamber, and other factors, the increase of flow rate of the secondliquid through the second port may vary. For example, in embodimentswhere the particles may be cells, the flow rate of the second liquidthrough the second port may be increased to between about 5 ml/min toabout 100 ml/min, such as about 10 ml/min to about 90 ml/min, about 15ml/min to about 85 ml/min, about 20 ml/min to about 80 ml/min, or evenabout 25 ml/min to about 75 ml/min. In other embodiments, the flow rateof second liquid through second port may be less than about 70 ml/min,less than about 65 ml/min, less than about 60 ml/min, less than about 55ml/min, less than about 50 ml/min, less than about 45 ml/min or evenless than about 40 ml/min. In other embodiments, the flow rate of secondliquid through second port may be greater than about 5 ml/min, greaterthan about 10 ml/min, greater than about 15 ml/min, greater than about20 ml/min, greater than about 25 ml/min, greater than about 30 ml/min oreven greater than about 35 ml/min.

In some embodiments, step 1340 may also be performed to treat theparticles. The second liquid may include some material that may be usedto modify the particles, or surround them with some predeterminedenvironment.

Flow 1300 then passes to step 1348 where liquid is removed through athird port at a lower force region. Referring to FIG. 14, step 1348 mayinvolve removing liquid through port 1436. It is noted that step 1348may be performed continuously throughout various steps of flow 1300. Forexample, as particles and liquid are introduced into the chamber at step1324, liquid may begin to be removed from the chamber through the thirdport. Also, as flow rates of liquid are increased, e.g., steps 1336,1344, liquid may be removed through the third port.

Flow 1300 passes from step 1348 to step 1352 where a second volume ofparticles and liquid are removed through the second port. Referring toFIG. 14, the second volume of particles and liquid may be removedthrough port 1440. Flow 1300 then ends at step 1356.

As noted above, flow 1300 may be utilized in processing any combinationof particles and liquid to concentrate, wash, and or treat particles. Inseveral embodiments, flow 1300 may be used to concentrate and wash cellsthat may be grown for research or therapeutic purposes. For example, inembodiments, a cell containing liquid may be generated from cells thatare grown in a liquid media. The cell containing liquid may be from acell expansion system. One example of a cell expansion system isdescribed in U.S. Pat. Nos. 8,309,347; and 8,785,181, which are herebyincorporated by reference in their entirety as if set forth herein infull. It is noted that the cells may be any type of cells, somenon-limiting examples including T-cells, mensenchymal stem cells,fibroblasts, red blood cells, leukocytes, etc.

In some embodiments, a volume of cells and liquid may be processed usingsome or all of the steps of flow 1300, in combination with chamber 1000(FIG. 10A-E). In embodiments, steps 1312, 1316, 1324, 1328, 1348, and1352 of flow 1300 may be performed.

Chamber 1000 may be rotated to subject the chamber to the centrifugalfield (e.g., steps 1312 and 1316). A volume of cells, e.g., T-cells, maybe introduced into the volume 1008 through port 1004 (e.g., step 1324).After the first volume of cells and liquid is introduced, a secondliquid may be introduced through port 1012 (e.g., step 1328) to maintainthe particles in suspension.

The centrifugal field may push cells toward exit port 1012. The cellsmay be concentrated because the liquid introduced into the chambervolume 1008 with the particles may flow out of the chamber through port1016 (e.g., step 1348). Step 1328 may ensure that as the particles arebeing concentrated, they do not pack or permanently agglomerate.Finally, at step 1352, a second volume of cells and liquid that is lessthan the first volume may be removed.

In other embodiments, not only are the cells concentrated, but they maybe additionally processed by being washed. In these embodiments,optional steps 1332, 1336, 1340, and 1344 may be performed in additionto steps 1312, 1316, 1324, 1328, 1348, and 1352.

Accordingly, after the first volume of cells and liquid is introducedinto chamber 1000, and second liquid is flowing into the chamber throughport 1012, (steps 1324 and 1328), step 1332 may be performed to change aviscosity of the liquid in volume 1008. This step may be performed byadding a second liquid, e.g., a wash liquid into volume 1008 through,for example, port 1004 (e.g., step 1336).

Step 1340 may be performed by adding the second liquid (e.g., increasingthe flow rate) into port 1012. The second liquid may flow through thebed of cells and out of the chamber through the port 1016. As notedabove, step 1332 is performed to change the viscosity of the liquid inthe chamber to allow the wash liquid introduced at step 1340 to displacethe liquid in the chamber and wash the liquid and any other materials inthe liquid out through the port 1016. The concentrated and washed cellsmay then be removed through the port 1012 at step 1352.

In some embodiments, a large range of volumes of cells and liquid (e.g.,first volume) may be processed using the steps of flow 1300. The volumesmay range from several hundred milliliters up to a hundred liters. Insome embodiments, flow 1300 may process (wash and concentrate) volumesof about 10 liters in about 60 minutes or less, such as a volume ofabout 5 liters in about 30 minutes or less, or a volume of about 3liters in about 20 minutes or less.

In some embodiments, flow 1300 (e.g., in combination with chamber 1000)may be performed to accomplish volume reductions of about 50%, about60%, about 70%, about 80%, about 90%, or even about 95%. Also, dependingon the conditions and other parameters, the cell loss, e.g., thedifference between the number of cells in the original volume and thenumber of cells in the final volume, may be less than about 10%, lessthan about 5%, less than about 4%, less than about 3%, less than about2%, less than about 1%, or even less than about 0.5%. That is, thevolume of cells in the second volume may include greater than about 85%of the cells in the first volume, greater than about 90% of the cells inthe first volume, greater than about 95% of the cells in the firstvolume, greater than about 98% of the cells in the first volume, greaterthan about 99% of the cells in the first volume, greater than about99.5% of the cells in the first volume, or even 99.9% of the cells inthe first volume.

It is noted that although the description above has been made withrespect to a batch process, where a volume of particles and liquid areprocessed (e.g., concentrated, washed, and/or treated), flow 1300 andthe chambers 500, 600, 700, 800, 900, and 1000, described above, mayalso be used in processes that are continuous.

As may be appreciated, in some embodiments, a volume of particles mayexceed the capacity of a chamber. In these embodiments, step 1352 may beperformed periodically in order to remove particles from the chamber.With step 1352 being performed periodically, a continuous process ofconcentrating particles may be performed. Removing the particles fromthe chamber may be performed using a number of techniques. For example,a pump may be used to pump particles out of the chamber volume.

In other embodiments, a continuous process may be implemented bymodifying a ratio of flow rate through two ports. Using chamber 1000 asan example, a ratio of the flow rate of liquid and particles throughport 1004 and a flow rate of liquid trough port 1012 may be used. Inother words, the flow rate though port 1012 may be reduced (or the flowrate through port 1004 may be increased) so that particles are pushedthrough port 1012 and into a pathway (e.g., tubing) in fluidcommunication with port 1012. In embodiments, a container or otherstorage volume may be connected in fluid communication with port 1012 toprovide additional volume for storing particles in a continuous process.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the methods and structure ofthe present invention without departing from its scope. Thus it shouldbe understood that the invention is not be limited to the specificembodiments or examples given. Rather, the invention is intended tocover modifications and variations within the scope of the followingclaims and their equivalents.

While example embodiments and applications of the present invention havebeen illustrated and described, it is to be understood that theinvention is not limited to the precise configuration and resourcesdescribed above. Various modifications, changes, and variations apparentto those skilled in the art may be made in the arrangement, operation,and details of the methods and systems of the present inventiondisclosed herein without departing from the scope of the claimedinvention.

What is claimed is:
 1. A method of processing particles, the methodcomprising: subjecting a chamber to a centrifugal field by rotating thechamber; introducing a first volume of particles and liquid into achamber volume through a first port of the chamber; after introducingthe first volume of particles and liquid into the chamber volum,introducing a second liquid into the chamber volume through a secondport of the chamber, wherein the second port is positioned in a higherforce region of the centrifugal field than the first port; removingliquid through a third port of the chamber, wherein the third port ispositioned in a lower force region of the centrifugal field than thefirst port; and removing a second volume of particles and liquid throughthe second port, wherein the second volume of particles and liquid issmaller than the first volume of particles and liquid.
 2. The method ofclaim 1, further comprising: after the introducing the first volume ofparticles and liquid and before removing the second volume of particlesand liquid, changing the viscosity of liquid in the chamber volume. 3.The method of claim 2, wherein changing the viscosity comprisesintroducing the second liquid into the chamber volume through the firstport.
 4. The method of claim 3, further comprising: after the changingthe viscosity, introducing a washing fluid into the chamber volume towash particles in the chamber volume.
 5. The method of claim 4, whereinthe washing fluid is introduced through the second port.
 6. The methodof claim 1, wherein the chamber volume is defined by a side wall, andwherein the first port is in a portion of the side wall that defines atrailing volume of the chamber volume.
 7. The method of claim 1, whereinthe introducing the second liquid through the second port maintainsparticles in the chamber volume in liquid suspension to reduce packingor agglomeration.
 8. A method of processing particles, the methodcomprising: subjecting a chamber to a centrifugal field by rotating thechamber; introducing a first volume of particles and liquid into achamber volume through a first port of the chamber; introducing a secondliquid into the chamber volume through a second port of the chamber,wherein the second port is positioned in a higher force region of thecentrifugal field than the first port; removing liquid through a thirdport of the chamber, wherein the third port is positioned in a lowerforce region of the centrifugal field than the first port; removing asecond volume of particles and liquid through the second port, whereinthe second volume of particles and liquid is smaller than the firstvolume of particles and liquid; and after the introducing the firstvolume of particles and liquid and before removing the second volume ofparticles and liquid, changing the viscosity of liquid in the chambervolume, wherein changing the viscosity comprises introducing the secondliquid into the chamber volume through the first port, and wherein thesecond liquid is introduced through the first port at a first flow rate,which is greater than a flow rate of the second liquid introducedthrough the second port.
 9. The method of claim 8, further comprising:after the changing the viscosity, increasing the flow rate of the secondliquid through the second port to a second flow rate.
 10. The method ofclaim 9, further comprising: after the changing the viscosity,decreasing the flow rate of the second liquid through the first port toa third flow rate.